Kawashima, DOI: et al., 10.4172/2157-7145.

1000269 J Forensic Res 2015, 6:1 

Research Article Open Access Estimating the Time after Death on the 
Basis of Corneal Opacity 
Wataru Kawashima*, Katsuhiko Hatake, Risa Kudo, Mari Nakanishi, Shigehiro Tamaki, Shogo Kasuda, Katsuya Yuui 
and Akiko Ishitani Department of Legal Medicine, Nara Medical University School of Medicine, 840 Shijo-cho, 
Kashihara, Nara 634-8521, Japan 
Abstract 
Estimation  of  the  time after death (TAD) is an important aspect of forensic science. The cornea becomes increasingly opaque 
with  an  increase  in TAD and corneal opacity is used for TAD estimation. However, previous methods are subjective, and there is 
a risk of human error. To establish an objective method, we propose a new method for TAD estimation. 
We  applied  RGB  image  analysis  to the corneal color of cadavers. We then examined if there was a correlation between these 
color  parameters  and  actual  TAD,  age,  position  at  death,  and  environmental  temperature.  We  found  that  corneal  opacity  was 
affected only by TAD. 
The method described here is objective and easy to use, and TAD can be estimated within a very short period of time, making 
this  method  particularly  useful.  To  further  increase  the  accuracy  of TAD estimation, other quantifiable parameters could also be 
evaluated. 
Keywords: Forensic science; Corneal opacity; Computer-assisted image analysis; Time after death; Autopsy; Cadaver 

Introduction 
During  an  autopsy,  accurate  estimation  of  the  time  after  death  (TAD)  is  very  important.  The  association  between  corneal 
transparency  and  TAD  was  established  in  1965  [1],  and  it  is  now  generally  accepted  in  the  forensic  community that the cornea 
becomes increasingly opaque with a longer TAD. Therefore, corneal opacity is an accepted means for estimating TAD. 
Although  the  use  of corneal opacity in estimating TAD has been established, there are limitations in its application. Owing to 
the  use  of  macroscopic  evaluation,  the  degree  of  opacity  is classified only as mild, moderate, or high. In addition, the evaluation 
criteria  used  to  determine  the  classification  rely  on  the  examiner’s  judgment;  therefore,  different  examiners  can  reach  different 
conclusions for the same cadaver. 
A  previously  used  method  of  TAD  estimation  based  on  corneal  opacity  is  the  gray  scale  method  [2], which estimates TAD 
through  comparison  of  the  degree  of  corneal  opacity  to  the  gray  scale,  but  this  method  also  relies  on  the examiner’s judgment. 
Other,  objective  means  of  TAD  estimation  are  also  available,  including  the  measurement  of  laser  light  transmittance  [3,4]  and 
corneal  thickness  [5],  calculation  of  the  number  of  dead  endothelial  cells in the cornea [6,7], and estimation of the state of DNA 
degradation in the corneal cells [8]. However, most of these methods require removal of the eyeball, which affects the appearance 
of  the  cadaver.  They  also  require  specialized  procedures  and  equipment  as  well  as  a  considerable  amount  of  time  for  TAD 
estimation. 
Image analysis is another option for TAD estimation. Zhou et al. [9] and Liu et al. [10] reported the use of image analysis as a 
TAD estimation method in mice and rabbits. They calculated the degree 
relationship  between  corneal  opacity  and  TAD  as  well  as  the  influence  of  several  factors  that  may  affect  the  degree  of  corneal 
opacity. Finally, we proposed an estimation method for TAD by evaluating the degree of corneal opacity. 

Materials and Methods 

The study protocol was approved by the ethics committees of the Nara Medical University. 
The  corneal  images  were collected during autopsies that were performed in the Department of Legal Medicine, Nara Medical 
University,  between  April  2011  and  December  2012.  Cadavers  (n=174) within 48 h after death and whose age, gender, and time 
of death were known or could be identified within a specific time range were used. And none had been stored in the freezer till 48 
h  after  death.  The  cadavers  with  conditions that could affect corneal opacity, such as fire damage, a high level of decomposition, 
and considerable damage of the craniocervical region, were excluded from the present study. 
During  the  autopsy  of  each  cadaver,  photographs of both eyes were taken using a digital camera (D3000; Canon, Japan). We 
positioned  the  camera  just  above  the  cadaver,  and  the  light  source  placed  slightly  left  of  and  above  it  in  order  to  avoid  the 
reflection  of  light  seen  on  the  cornea  when  taking  photograph.  To  compare  the  degree  of opacity under different lighting, 54 of 
the  174  cadavers’  corneas  were  photographed under light from light emitting diodes (LEDs) as well as room light (Figure 1a and 
1b). The time from the known time of death to the time that the photograph was taken was defined as the actual TAD. 
*Corresponding author: Wataru Kawashima, Department of Legal Medicine, Nara Medical University School of Medicine, 840 
Shijo-cho, Kashihara, Nara 634-8521, Japan, Tel: +81-744-29-8843; Fax: +81744-29-1116; E-mail: watack14@naramed-u.ac.jp 
of corneal opacity on the image according to the TAD and found a correlation between the degree of opacity and TAD; the 
degree 
Received January 07, 2015; Accepted January 25, 2015; Published January 30, 2015 of corneal opacity increased with increasing 
TAD. However, this technique has not been used for human corneas. 
Citation: Kawashima W, Hatake K, Kudo R, Nakanishi M, Tamaki S, et al. (2015) Estimating the Time after Death on the Basis 
of Corneal Opacity. J Forensic Res 6: In order to use image analysis for the evaluation of corneal 
269. doi:10.4172/2157-7145.1000269 
opacity  in  humans,  we devised a simple and objective method for photographing the corneal color of cadavers during an autopsy. 
Following the use of this newly developed method, we evaluated the 
Copyright:  ©  2015  Kawashima  W,  et  al.  This  is  an  open-access  article  distributed  under  the  terms  of  the  Creative  Commons 
Attribution  License,  which  permits  unrestricted  use,  distribution,  and  reproduction  in  any  medium,  provided  the original author 
and source are credited. 
J Forensic Res ISSN: 2157-7145 JFR, an open access journal 
Volume 6 • Issue 1 • 1000269 
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ISSN: 2157-7145 

Journal of Forensic Research 

 
Citation:Kawashima W, Hatake K, Kudo R, Nakanishi M, Tamaki S, et al. (2015) Estimating the Time after Death on the Basis 
of Corneal Opacity. J 
Forensic Res 6: 269. doi:10.4172/2157-7145.1000269 
Page 2 of 5 
There  were  positive  correlations  between  corneal  color,  as  estimated  from  RGB  shading,  and  actual  TAD  for  both  light 
sources  (Table  1, Figure 3a and 3b), and the degree of corneal opacity increased with increasing TAD. The t-test results indicated 
a  significantly  larger  mean  RGB  value  in  the  presence  of  LEDs  than  that  in  room  light  over  the  entire  time  period  and in each 
TAD 10-h category, except for the category of 40-48 h (Figure 4). 
Significant positive correlations were also observed between TAD and corneal color, defined using RGB shading, in each 
group classified 
Figure 1: (a and b) Images of cadaveric corneas under room light (a) and LED (b) for the analysis of corneal opacity LED: Light 
Emitting Diode. 
by age, position at death, and environmental temperature (Table 2). The correlation between TAD and RGB shading increased 
with increasing age; however, the correlations for environmental temperature and Corneal Color Diagnosis 
position at death were similar across all categories. The results of the 
From the photographic images, corneal color was defined using RGB shading in an open-source software program (Image J, 
http:// imagej.nih.gov/ij/index.html). In this program, every color is defined as combinations of shades of red (R), blue (B), and 
green (G), from black (R, B, G=0, 0, 0) to white (R, B, G = 255, 255, 255). We chose 5 points of the corneal region (center, top, 
bottom, right side, left side) for analysis, and the average values for each cornea were used in subsequent analyses (Figure 2). 
ANCOVA revealed no relationship between corneal opacity and each factor (age, position at death, and environmental 
temperature). 
Based on these results, proposed equations for estimating TAD using corneal color analysis are provided in Table 3. No 
parameter had a VIF ≥10, allowing for the inclusion of all parameters in the regression model. Although age was not a significant 
factor in the ANCOVA results, it was included in the equations because of the correlations between increasing age and greater 
corneal opacity. Using these equations, the differences between the estimated and actual Statistical Analysis 
TAD, referred to as the mean error, were found to be 6.3 (4.1) h for 
All  data  were  analyzed  using  SPSS  v19.0  (IBM  Corp,  Armonk,  NY).  Age  is  expressed  as  mean  (SD)  values.  Statistical 
significance was set at a two-tailed P value of <0.05. 
the  room  light  condition  and  9.7  (7.6)  h  for  the  LED  condition  (Table  4).  The  mean  error  of  the  estimation  in  the  room  light 
condition was significantly less than that of the estimation in the LED condition. 
Pearson  correlation  coefficient  analyses  were  conducted  to  determine relationships between TAD and corneal color (defined 
as RGB shading) under both room light and LEDs. The actual TAD values 
In the room light condition, the mean error was within 5 h of the actual TAD in 50% of the estimated TAD values (Figure 5). 
Further assessment of the mean errors showed that 31 cases had mean errors >10 h, and these 31 cadavers were all aged over 60 
years. were grouped according to 10-h intervals (0-9, 10-19, 20-29, 30-39, and 40-48 h). These groups were used to conduct 
student t-tests between the results obtained in room light and those obtained using LEDs, to investigate the differences in the 
degree of corneal opacity in both 
When  comparing  the  TAD  10-h  categories,  the  mean  errors  of  the categories >10 h and <30 h were each significantly lower 
than those of the other TAD categories (Figure 6). 
lighting sources. 
Discussion We classified the cadavers into 6 age groups 
(<40, 40-49, 50-59, 60- 
The results of this study demonstrate the 
usefulness of computer- 69, 70-79, and ≥80 years), 5 positions at death (dorsal, prone, lateral, 
based image analysis in the estimation of TAD during 
an autopsy. The hanging, and sitting), and 3 environmental temperature groups (<10°C, 10-20°C, and ≥30°C) to investigate the 
effects of age, position at death, and environmental temperature on corneal opacity; these effects were analyzed using an 
ANCOVA. The Pearson correlation coefficient was also determined for TAD and corneal color in each group. 
Multiple  regression  model  analysis  was  conducted  to  determine  the effect of the variables that influenced corneal opacity on 
TAD  estimation;  from  this,  we  proposed  equations  for  estimating  TAD  using  RGB  shading  under  room  light  and  LEDs. 
Multi-collinearity was assessed using the variance inflation factor (VIF) for each parameter prior to the regression analysis. 
We  performed  data  splitting  for  cross-validation  of  the  model.  The  calculated  TAD  estimates  were  then  compared  to  the 
recorded  actual  TAD  of  the  cadavers;  the  calculated  differences  between  the  estimated  and  actual  TAD  are  referred  to  as  the 
mean error. 
Correlation coefficient (r) Room light LED P value TAD & R 0.60 0.35 <0.01 Results 
TAD & G 0.60 0.40 <0.01 TAD & B 0.56 0.42 <0.01 Eyes from 104 male and 70 female cadavers (average age, 63.9 [18.1] 
years and 68.9 [21.9] years, respectively) were evaluated in this study. The average TAD was 20.0 (9.3) h (range: 3-48 h). 
Table  1:  Relationships  between  TAD  and  corneal  color  (defined  as RGB shading) under 2 light conditions-room light and LED. 
R: Red; B: Blue; G: Green; TAD: Time after Death; LED: Light Emitting Diode. 
J Forensic Res ISSN: 2157-7145 JFR, an open access journal 
Volume 6 • Issue 1 • 1000269 
Figure 2: Five points of the corneal region used for the calculations of the average RGB shading to determine corneal opacity. 
 
Citation:Kawashima W, Hatake K, Kudo R, Nakanishi M, Tamaki S, et al. (2015) Estimating the Time after Death on the Basis 
of Corneal Opacity. J 
Forensic Res 6: 269. doi:10.4172/2157-7145.1000269 
Page 3 of 5 
Figure 3: (a and b) Correlations between time after death (TAD) and corneal color (defined as RGB shading) in the room light 
condition (a) and LED condition (b) R: Red; B: Blue; G: Green; LED: Light Emitting Diode. 
Figure 4: Comparison of the mean RGB value under room light and LED over the entire time period and in each 10-h category. 
** P<0.01, comparison between room light and LED. R: Red; B: Blue; G: Green; LED: Light Emitting Diode. 
J Forensic Res ISSN: 2157-7145 JFR, an open access journal 
Volume 6 • Issue 1 • 1000269 
 
Citation:Kawashima W, Hatake K, Kudo R, Nakanishi M, Tamaki S, et al. (2015) Estimating the Time after Death on the Basis 
of Corneal Opacity. J 
Forensic Res 6: 269. doi:10.4172/2157-7145.1000269 
Page 4 of 5 
TAD&R P value TAD&G P value TAD&B P value 
Age category (years) 
<40 0.43 <0.01 0.41 <0.01 0.36 <0.01 40-49 0.44 <0.01 0.44 <0.01 0.44 <0.01 50-59 0.62 <0.01 0.54 <0.01 0.47 <0.01 
60-69 0.61 <0.01 0.57 <0.01 0.52 <0.01 70-79 0.69 <0.01 0.64 <0.01 0.58 <0.01 ≥80 0.65 <0.01 0.73 <0.01 0.75 <0.01 Position 
at death 
Dorsal 0.61 <0.01 0.61 <0.01 0.56 <0.01 Prone 0.61 <0.01 0.57 <0.01 0.52 <0.01 Lateral 0.69 <0.01 0.66 <0.01 0.66 <0.01 
Hanging 0.5 <0.01 0.51 <0.01 0.52 <0.01 
Sitting 0.65 <0.01 0.69 <0.01 0.7 <0.01 
Environmental temperature (°C) 
<10 0.61 <0.01 0.61 <0.01 0.56 <0.01 10-20 0.61 <0.01 0.57 <0.01 0.52 <0.01 ≥30 0.69 <0.01 0.66 <0.01 0.66 <0.01 
Table 2: The correlation coefficients for the relationships between TAD and corneal color (defined as RGB shading) displayed as 
groups by age, position at death, and environmental temperature of the cadaver. R: Red; B: Blue; G: Green; TAD: Time after 
Death. 
Room light LED TAD =-0.06 age + 0.2R- 0.01G + 0.13B + 29.5 TAD = -0.17 age - 0.005R - 0.33G + 0.6B + 69.9 R 0.69 0.81 
R2 0.47 0.66 
Adjusted R2 0.45 0.62 
Table 3: Equations for the estimation of TAD using photographic corneal color evaluation in shades of R, B, and G under 2 
different light conditions (room light and LED). R: Red; B: Blue; G: Green; TAD: Time after Death; R: multiple correlation 
coefficient; R2: coefficient of determination; LED: Light Emitting Diode. 
Room light condition LED condition TAD (h) Mean error (h) S.D. N Mean error (h) S.D. N 
0–9 8.9 3.5 27 9.9 7.6 14 10-19 4.5 3.1 52 7 6.5 12 20-29 4.5 2.7 51 11.2 7.8 16 30-39 10.2 5 27 10.1 8.6 6 40-48 10.2 5.5 17 
11.1 14.8 6 
All 6.3 4.1 174 9.7 7.6 54 
Table 4: The mean error of the TAD equation, compared to actual TAD, when using the corneal color analysis from photographs 
taken in 2 different light conditions (room light and LED). S.D., standard deviation; TAD: Time after Death; LED: Light 
Emitting Diode. 
positive relationships that existed between RGB shading in the cornea 
previous studies were taken under room light; the 
effect of the light and actual TAD indicated that the cornea became opaque (higher 
source on corneal opacity was not taken into 
consideration. In this RGB score) with increasing TAD. These results support those reported 
study, LEDs resulted in a higher degree of corneal 
opacity than room previously by Aoki [1]. 
light for the TAD 10-h categories <40 h. The light produced by LEDs 
Computer-based  image analysis offers the benefit of digitization, which confers many advantages such as stability, reliability, 
non-  invasiveness,  reproducibility,  and the elimination of human error. In this study, we adapted the image analysis methodology 
for  humans  by  using  the  RGB  score  to  calculate  the  degree  of  opacity.  Because  the  RGB  score  can  indicate  the  color  of  the 
cornea through the choice of specific points on the cornea, it improves the objectivity, simplicity, 
 may be brighter than room light, resulting in larger values for the RGB score and a more opaque corneal color. Moreover, the 
slight change in corneal color with TAD was not apparent under LEDs, as indicated by the smaller correlation coefficient with 
LEDs than that with room light. In addition, the mean error of the equation developed with room light was significantly smaller 
than that of the LED equation, leading us to believe that room light is more acceptable for this method of TAD estimation. and 
speed of the analysis. Moreover, the RGB score reflects an objective evaluation of opacity, further eliminating the risk of human 
error while, at the same time, providing a segmentalized numerical score comprised of each color (R, G, B) on a scale of 0 to 
255. 
Corneal opacity can also occur due to transpiration of fluid and degeneration of endothelial cells and may therefore be 
accelerated in high temperatures. Corneal opacity may also be affected by postmortem hypostasis that occurs when the person is 
in a prone position at the Despite the superiority of image analysis over other methods for 
time of death. Age is another factor to consider, as it is 
generally estimating TAD, several factors affecting the quality of the photograph 
accepted that corneal opacity increases with age due 
to a decrease in and the condition of the cornea should be considered. Photographs in 
the number of corneal endothelial cells, which results in an increase 
J Forensic Res ISSN: 2157-7145 JFR, an open access journal 
Volume 6 • Issue 1 • 1000269 
 
Citation:Kawashima W, Hatake K, Kudo R, Nakanishi M, Tamaki S, et al. (2015) Estimating the Time after Death on the Basis 
of Corneal Opacity. J 
Forensic Res 6: 269. doi:10.4172/2157-7145.1000269 
Page 5 of 5 
significantly  larger.  The  shorter  time  period  would  result  in  very  little  change  in  corneal  opacity,  while  the  longer  time  period 
would  result  in  a  large  change  in  corneal  opacity.  The  cadavers  with  mean  errors  >10  h  were  all aged over 60 years, indicating 
that care should be taken when using this method in older people. 
The results of our image analysis, an objective method, confirm that corneal color increases in opacity with longer TADs. 
Autopsies performed within 10-30 h of death can confidently use this method for accurate estimation of TAD. However, with 
shorter and longer TAD, the accuracy may diminish. Despite these limitations, this estimation method is the first to support the 
use of image analysis to determine corneal color. Thus, it may be a useful alternative. As the use of LEDs Figure 5: The mean 
error of the TAD equation, compared to the actual TAD, when 
becomes more common, it will be necessary to 
develop a more accurate using the corneal color analysis from photographs taken in room light. TAD: Time After Death. 
method for TAD estimation under LEDs. 
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Figure 6: Comparison of the mean error in the estimation of TAD, compared to the actual TAD in each of the 10-h TAD 
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postmortem interval in rabbits. J Huazhong 
In  this  study,  we also developed equations to estimate TAD from RGB image analysis in room light. Although the total mean 
error of the room light equation was considered to be small (6.3 [4.1] h), the 
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errors at the shorter (<9 h) and longer (>30 h) TAD times were 
J Forensic Res ISSN: 2157-7145 JFR, an open access journal 
Volume 6 • Issue 1 • 1000269